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ARTICLE IN PRESSG ModelRBI-10496; No. of Pages 8

Process Biochemistry xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Process Biochemistry

jo ur nal home p age: www.elsev ier .com/ locate /procbio

tudy on dyeing wastewater treatment at high temperature by MBBRnd the thermotolerant mechanism based on its microbial analysis

hao Li a,b, Zhen Zhang b, Yi Li a,b, Jiashun Cao a,b,∗

Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education, Hohai University, Nanjing 210098, ChinaCollege of Environment, Hohai University, Nanjing 210098, China

r t i c l e i n f o

rticle history:eceived 23 June 2015eceived in revised form 3 August 2015ccepted 9 August 2015vailable online xxx

eywords:yeing wastewaterBBR

a b s t r a c t

The dyeing wastewater treatment under increasing high temperature condition was studied in MBBRs.Results showed that as the temperature increase, the COD removal exhibited two best performancesat 40 ◦C and 50 ◦C, and the maximum NH3-N removal was obtained at 35 ◦C and 40 ◦C. The optimumtemperature for extracellular polymeric substances (EPS) yield is 45 ◦C and the humic acid was the majorcontributor. Microbial community was analyzed by the association of DGGE, real-time PCR and high-throughput sequencing technologies. The highest abundance of AOB (characterized by amoA genes) wasobtained at 35 ◦C, and it confirmed that the biofilm (attached on fillers) can facilitate the AOB abundancemaintaining in the sludge. The “precursor and successor” thermophilic communities were enriched and

◦ ◦
icrobial community
PShermotolerant mechanism

dominant at different temperature stages, which mainly conclude genera Caldilinea (from 35 C to 45 C)and genera Rubellimicrobium and Pseudoxanthomonas (over 50 ◦C), respectively. It meant the thermophiliccommunity displayed great evolution at the critical temperature “45 ◦C|50 ◦C”. Additionally, the processof thermotolerant mechanism establishment of the sludge in the MBBRs is proposed and the “two-stageenrichment” of different thermophilic communities was considered as the key procedure.

© 2015 Elsevier Ltd. All rights reserved.

. Introduction

The textile industry is one of the most important industrieshose produced wastewater is characterized by high COD, high

olor and refractory organics [1]. The biological technologies, ratherhan some physical and chemical treatments [2], are considered ashe most widely applicable method for dyeing wastewater treat-

ent, because it is simple and cost-effective [3]. Most of theresent biological treatment for dyeing wastewater operated underesothermal condition (below 40 ◦C). However, the discharge of

extile wastewater usually suffers from relative high temperature.or example, the water temperature of pulp and bleaching effluentor knitted products is about 40–45 ◦C, and the desizing scouring

Please cite this article in press as: C. Li, et al., Study on dyeing wastewatemechanism based on its microbial analysis, Process Biochem (2015), h

astewater for woven fabric can reach 50 ◦C or even higher.The dyeing wastewater with high temperature requires cool-

ng prior to biological proceeding, or would greatly impact on the

Abbreviation: MBBR, moving bed biofilm reactor; COD, chemical oxygenemand; EPS, extracellular polymeric substances; DGGE, denaturing gradient gellectrophoresis; PCR, polymerase chain reaction; AOB, ammonia oxidizing bacteria.∗ Corresponding author at: College of Environment, Hohai University, Nanjing10098, China. Fax: +86 2583786701.

E-mail address: [email protected] (J. Cao).

ttp://dx.doi.org/10.1016/j.procbio.2015.08.007359-5113/© 2015 Elsevier Ltd. All rights reserved.

effluent [4,5]. The cooling process would increase the cost and thecooling equipment corrosion cannot be ignored. So, there is a dearthof knowledge on dyeing wastewater treatment under the highertemperature.

Thermophilic bacteria had been widely found [6] and used forwastewater treatment years ago, such as brewery wastewater [7],slaughter wastewater [8] and vegetable waste [9]. Comparing to themesothermal, thermophilic treatment possesses own higher pollu-tant degradation rates, with about 3–10 times higher [4]. Moreover,thermophilic treatment possesses also combine with other bene-fits (e.g., lower excess sludge production [10], or higher pathogenicbacteria removal [11]).

However, the wastewater at high temperature has to facenew challenges in biological treatment. High temperature leads topoor sedimentary property of the sludge, which is usually com-pensated by biofilm system, in order to reduce the sludge loss[12]. Another challenge is the severe stress for the microorgan-ism growth. Although the research on microbial community earnedmuch attention at the high temperature [13], since microorganismsplay the key role in biological treatment, the establishment process

r treatment at high temperature by MBBR and the thermotolerantttp://dx.doi.org/10.1016/j.procbio.2015.08.007

of thermotolerance mechanisms (as the temperature increase), isstill lack of systematic study.

In this study, the moving bed biofilm reactor (MBBR) was appliedto treat dyeing wastewater, with the additional fillers to help com-

dx.doi.org/10.1016/j.procbio.2015.08.007


http://www.sciencedirect.com/science/journal/13595113

http://www.elsevier.com/locate/procbio

mailto:[email protected]


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ARTICLERBI-10496; No. of Pages 8

C. Li et al. / Process Bioc

ensate for the poor sedimentary property of the sludge. Under theerobic condition, as the temperature increasing gradually from0 ◦C to 55 ◦C in the MBBR, we aim to: (1) investigate the pollutantsemoval efficiencies in the dyeing wastewater at different tempera-ure stages; (2) compare the microbial communities at temperaturetages, and propose the process of thermotolerance mechanismstablishment in the sludge as the temperature increasing grad-ally.

. Material and method

.1. Reactor and operation

The moving bed biofilm reactors (MBBR) were constitutedy polymethyl methacrylate tanks (active volume 2 L), with theolypropylene suspended filler (K1-type) addition in order tonhance the flocculation ability of sludge. In order to ensure theepeatability, two identical MBBR reactors operated in parallelThe results were obtained by averaged value of the two). Each

BBR operated continuously, better agreed with the practicalpplication. The hydraulic retention time (HRT) of the reactorsas fixed at 24 h and the dissolved oxygen (DO) was maintained

t 2.0 ± 0.4 mg/L during the whole experiment, which conformedo (simulated to) the practical process of the WWTP. The initialludge retention time (SRT) was about 15 days and the initial mixediquor suspended solids (MLSS) was 12,500 mg/L (the MLSS woulde reduced when the temperature increased over 35 ◦C, Figure2).

The temperature was gradually increased stage by stage,nclude 30 ◦C (the original inoculated sludge), 35 ◦C, 40 ◦C,5 ◦C, 50 ◦C and 55 ◦C. Each stage was kept for at least 15ays after both the two MBBRs’ performance were stablecharacterized by COD removal efficiency according to the efflu-nt), and then the water temperature was increased to nexttage by 1 ◦C/day, avoiding the shock of abrupt temperaturehange.

.2. Characterization and analysis of sludge properties

The raw wastewater was derived from the effluent of a dye-ng company whose products majored in cotton woven fabricnd a part of jean (which leaded to high azo dyestuffs andndigo dye in the wastewater), and was diluted (2-fold dilu-nt) to COD 650 ± 80 mg/L and NH3-N 18 ± 2.2 mg/L (the rawastewater was characterized by Table S1). The diluted influ-

nt with relative low NH3-N concentration (also the reactorsere equipped with covers) meant to prevent much volatiliza-

ion of ammonia gas at high temperature by aerating (i.e. airift effect). The performance of the reactors was monitoredvery day, including COD and NH3-N, according to the Standardethods [14]. The obtained data were averaged by the two reac-

ors.

.3. Characterization of extracellular polymeric substances (EPS)f the sludge

The EPS of the sludge at each temperature gradient (for sus-ended sludge only) was investigated. The EPS extraction processas performed according to the protocol described [15], including

oluble EPS, loosely bound EPS and tightly bound EPS. The pro-ein and carbohydrates contents were determined referred to therevious reports [16].


Excitation-emission matrix (EEM) fluorescence spectroscopyas further used to analyze the EPS by the fluorescence spec-

rophotometer (Hitachi, F7000), which includes the followinguorescence techniques: (i) excitation (EX) and emission (EM) were

PRESStry xxx (2015) xxx–xxx

both fixed at 200–600 nm, (ii) synchronous scan at a constant off-set wavelength 5 nm between excitation and emission. The contourmap was obtained by Origin 8.0.

2.4. Microbial community analysis

The sludge samples at the end of each temperature stage werecollected (300 mg) and the total genomic DNA was extracted fromeach sample by using the FastDNA kit (Q-Biogene, MP Biomedicals,UK) as described in the manufacturers’ instructions. The extractedDNA samples were stored at −20 ◦C, preparing for the followingPCR-DGGE, real time PCR and High-throughput sequencing analy-sis.

The PCR of 16S ribosomal DNA genes (V3 region, short for 16Sgenes) was performed for the total eubacteria investigation withthe primers 338f (5′-CCTACGGGAGGCAGCAG-3′, with GC-clampbefore DGGE) and 518 r (5′-ATTACCGCGGCTGCTGG-3′), under thefollowing conditions: 94 ◦C/5 min denaturation step; 30 cyclesof 94 ◦C/30 s, 58 ◦C/30 s, 72 ◦C/45 s; and a final extension step at72 ◦C/10 min.

DGGE was carried out in a denaturing gradient gel elec-trophoresis system for the purified products of PCR. Polyacrylamidegels (10% (w/v)) were 18 cm × 18 cm, thickness of 0.75 mm. Theelectrophoresis was conducted in 1 × TAE buffer at 60 ◦C. Thecondition of electrophoresis was 100 V, 12.5 h with denatur-ing gradients of 35–65%. Gels were photographed using Kodak1D Image Analysis Software after stained by ethidium bromide(EB).

Quantification of AOB (nitrifying bacteria) was character-ized by amoA genes targeted real-time PCR, with the primersamoA-1F: GGGGTTTCTACTGGTGGT, amoA-2R: CCCCTCKGSAAAGC-CTTCTTC [17]. The real-time PCR condition was: 900 s at 95 ◦C,40 cycles of 15 s at 95 ◦C, 30 s at 63 ◦C, 30 s at 72 ◦C (alsofor data acquisition step). While approximative quantificationof the total eubacteria amount was performed by 16S genes,in order to act as the reference with the condition: 15 sat 95 ◦C, 30 s at 60 ◦C for 40 times of repetition. One laststep from 60 to 95 ◦C with an increase of 0.2◦/s was addedto obtain a specific denaturation curve to ensure the accu-racy of the amplification for all the real-time PCR assaysabove.

Plasmids (pEASY-T1Cloning Kit, Transtaq) containing cloned16S genes or amoA genes which were cloned to DH5� wereused to draw standard curves (r2 = 0.99 with amplificationefficiency 100.5% for 16S genes; r2 = 0.997 with amplifica-tion efficiency 97.1% for amoA genes). All the abundance dataof these genes were based on averaged 3 identical sam-ples.

High-throughput sequencing of the 16S genes (Miseq) wasconducted for further analysis of the sludge samples at each tem-perature stage after 16S rRNA gene PCR amplification and PCRproducts purification (for suspended sludge only). To amplify andsequence the V1V2 hypervariable region of the 16S rRNA gene,forward primer (50-AGAGTTTGATYMTGGCTCAG-30) and reverseprimer (50-TGCTGCCTCCCGTAGGAGT-30) were selected and dif-ferent 8-bases barcodes and a Guanine were linked to the 50end of each primer. Then the purified products were sent forsequencing using Illumina sequencing platform (Miseq, IlluminaInc., USA). The acquired data was processed by Sickle and Mothurprogram to remove the low quality of sequence and reduce noises


(according to Q-score >25). Finally, the filtered sequences wereassigned to a taxon by the RDP classifier. Additionally, the heatmapsand cluster analyses were obtained using the R program (R-3.1.0).


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ig. 1. Performance of COD and NH3-N removal during the whole experiment ashe temperature rise.

. Result and discussion

.1. Performance of the MBBRs and the temperature effectnalysis

.1.1. Performance of the MBBRs at different temperature stagePerformance of the MBBR reactors (averaged data of the two),

uring the whole experiment was obtained in Fig. 1, includingOD and NH3-N removal efficiency which we mainly focused on.The average COD and NH3-N concentration of the influent were50 mg/L and 18 mg/L, respectively.)

Fig. 1 shows the COD and NH3-N removal efficiencies in theBBRs at each temperature stage (from 35 ◦C to 55 ◦C, and the 30 ◦C

s the background value in the parent reactor for inoculation). Thetable COD removal efficiency and lasting for 15 days was defineds the end of each temperature stage.

According to Fig. 1, with the stepped increased temperaturerom 35 ◦C to 55 ◦C, the COD removal efficiencies (the average valuef platform period) were 60.7%, 63.2%, 69.8%, 54.2%, 70.1% and 41.5%or 30 ◦C, 35 ◦C, 40 ◦C, 45 ◦C, 50 ◦C and 55 ◦C, respectively. The CODemoval first increased gradually, and then decreased at 40 ◦C|45 ◦C,ut ascended again at 45 ◦C|50 ◦C. Finally, it dropped to very low

evel over 50 ◦C (further data over 55 ◦C was not shown).Obviously, in this study, higher temperature (such as 40 ◦C and


0 ◦C) can achieve relative higher COD removal, differing from somerevious study which stated COD removal would decrease as tem-erature rising [18]. It is because higher temperature usually can

PRESStry xxx (2015) xxx–xxx 3

promote metabolism rate which enhances the COD removal [4], andalso can enrich thermophilic bacteria during long time of acclimati-zation, which further contributes to the COD removal under highertemperature.

It is noticed that the two maximum removals 69.8% and 70.1%were obtained at 40 ◦C and 50 ◦C, but poor performance at theintermediate 45 ◦C (Fig. 1). It supposed that the thermotolerantmicroorganisms which were responsible for COD removal before40 ◦C was eliminated gradually when the temperature furtherincrease. But the new thermotolerant community achieved enrich-ment when the temperature increased to 50 ◦C. It is an indicationthat the microbial community (responsible for COD removal) hasgone through great changes from 40 ◦C to 50 ◦C (see the discus-sion on microbial community below). Correspondingly, the NH3-Nremoval efficiencies (the average value of the whole period at eachtemperature stage) were 33.3%, 39.1%, 38.5%, 28.0%, 17.5% and11.5%, respectively.

The relative low level of NH3-N removal in this study proba-bly resulted from the complex nitrogenous pollutants in dyeingwastewater [19]. As seen in Fig. 1, higher temperature greatlyaffected the nitrifying bacteria activity, for the nitrifying ability isalmost prevented over 42 ◦C according to previous study [20]. Theremained NH3-N removal over 50 ◦C was probably caused by theair lifting effect [21], even thought it has been prevented furthest inthis study (lower NH3-N concentration, cover equipped and relativelow aerating).

The profile of NH3-N removal exhibited first increment and thendeclination, and the maximum was obtained at 35 ◦C, with a sharpdecrease at 40–45 ◦C, which agreed with the theoretical optimaltemperature 35 ◦C of AOB activity [20]. However, at 40 ◦C, the AOBmaintained the nitrifying ability to a certain extent, which probablyresulted from the protective effect by some thermophilic bacteriaenrichment.

3.1.2. Temperature effect analysisTemperature is an important biological factor and affects many

biological processes. To better evaluate the (high) temperatureeffect on the reactor performance, the temperature coefficient (Q10)which is a reaction parameter reflecting the biological metabolicrate variation by temperature effect in the bio-reactors, wasapplied. Q10 is characterized by the rate of change in a biologicalsystem as a consequence of increasing the temperature by 10 ◦C[22]:

Q10 =(R1

R2

) 10(T2−T1)

(1)

where R is the rate (here refers to COD or NH3-N removal rate) andT is the temperature.

The relationship between biological degradation of pollutantsand temperature, characterized by Q10, were calculated by Eq.(1): 0.92, 0.82, 1.66, 0.60 and 2.85 for each temperature interval(30 ◦C|35 ◦C, 35 ◦C|40 ◦C, 40 ◦C|45 ◦C, 45 ◦C|50 ◦C, 50 ◦C|55 ◦C), while0.72, 1.03, 1.89, 2.56 and 2.31 for NH3-N removal. In the generalview, the value of Q10 indicates whether a biological metabolicreaction is sensitive to the temperature fluctuation. Therefore, theNH3-N removal was more sensitive to temperature, rather thanCOD removal, based on their Q10 comparison. However, it is alsoan indication that the biological process of COD removal can bet-


mechanism establishment of the sludge. Only over 50 ◦C, the CODremoval was easily affected by temperature (Q10 = 2.85 from 50 ◦Cto 55 ◦C).


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Fig. 3. The DGGE profile of each sample at different temperature stages. (“O”referred to the original suspended sludge at around 30 ◦C, “S” and “B” referred tosuspended sludge and biofilm attached on the fillers, respectively).

Table 1The closest species and their classification of the representative DGGE bands.

No. The closest species* Classification

B1 Caldilinea aerophila DSM 14,535 ChloroflexiB2 Oscillibacter valericigenes Sjm18-20 FirmicutesB3 Caldilinea tarbellica D1-25-10-4T ChloroflexiB4 Bacillus sp. 1NLA3E FirmicutesB5 Nitrosomonas eutropha C91 BetaproteobacteriaB6 Zunongwangia profunda SM-A87 BacteroidetesB7 Leptothrix cholodnii SP-6 BetaproteobacteriaB8 Acidothermus cellulolyticus 11B ActinobacteriaB9 Geobacillus thermoglucosidasius C56-YS93 FirmicutesB10 Gramella forsetii KT0803 BacteroidetesB11 Pseudoxanthomonas taiwanensis CB-225 FirmicutesB12 Paracoccus denitrificans PD1222 Alphaproteobacteria

ig. 2. The EPS composition analysis at each temperature stage from 30 ◦C to 55 ◦CThe “O” refers to the inoculated sludge at around 30 ◦C).

.2. EPS characterization and sludge properties analysis

The EPS of the sludge samples at the 6 temperature gradi-nts were characterized for analyzing the properties of the sludgeFig. 2).

The total amount of EPS (the total of soluble EPS, loosely boundPS and tightly bound EPS) was focused, and it first increased andhen decreased, the maximum value 1748 mg/L was obtained at5 ◦C. The component of EPS in the sludge mainly included extracel-

ular carbohydrate, protein and humic acid. The amount of proteinnd humic acid exhibited the similar variation (Fig. 2), among ofhich, the humic acid was dominant and took the main responsi-

ility of the total EPS amount change.The detailed variation of EPS component can be obtained

rom the EEM fluorescence spectroscopy at Fig. S1, which furtherncluded the different fraction of EPS (i.e. soluble EPS, loosely boundPS, and tightly bound EPS), demonstrating the EPS variation exhib-

ted consistency with the Fig. 2.The EPS synthesis is considered as a self-protection of microor-

anisms under stress condition [23], including high temperature24]. In this study, the high temperature (when below 45 ◦C) wouldtimulate the EPS production, especially for the increasing of therotein and humic aicd. It probably also resulted from the enrich-ent of some EPS high yield bacteria [25] (discussed below).owever, when the temperature further increased (higher than5 ◦C), it had an inhibitory effect on the production rates of allractions of EPS, or even damaged the cells.

Previous studies demonstrated that the optimum (relativeigher) temperature was 35–40 ◦C for some specific microorgan-

sms on producing highest amount of EPS [24,26]. Nevertheless, inhis study, 45 ◦C was the optimum temperature for EPS produc-ion. It is an indication that the stimulated EPS production was anntegrated effect of the whole microbial community, which wouldncrease the thermostability (by more EPS production) for the bio-ogical sludge. In fact, there is even higher optimal temperature55 ◦C) for the active sludge EPS production [27].

.3. Microbial community analysis comparison at differentemperature

.3.1. PCR-DGGE analysis of microbial communityAt the end of each temperature stage (the stable COD removal


fficiency was obtained and lasted for 15 days), the microbial com-unity in the sludge was considered to reach the homeostasis

or approach steady state) [28], and the sludge sampled to beicrobial analyzed. The microbial community was first analyzed

B13 Geobacillus thermodenitrificans NG80-2 FirmicutesB14 Rubellimicrobium thermophilum C-lvk-R2A-2T Alphaproteobacteria

by PCR-DGGE, for both suspended sludge and biofilm (on fillers)samples from 35 ◦C to 55 ◦C (together with the original inoculatedsuspended sludge, 30 ◦C)

According to Fig. 3, great difference was observed in the micro-bial community structures among these samples, especially for thecomparison among different temperature stages, which indicatedthe temperature effect took more responsibility in microbial com-munity variation, rather than the fillers effect. Additionally, at sametemperature stage, the biofilm presented more detectable bandsthan the suspended sludge (Fig. 3), which meant the fillers enabledthe stratified structure of the sludge (biofilm formation) and morediverse microbial community was enriched.

Some representative bands were cut and sequenced for analyz-ing the characteristic microorganisms (listed in Table 1).

B1 and B3 were identified as Caldilinea aerophila and Caldilineatarbellica, belonged to the thermophilic genus Caldilinea, which canuse various carbohydrates [29]. These two bands exhibited specificintense at 40 ◦C and 45 ◦C, but suddenly fainted (even disappear)over 50 ◦C according to Fig. 3, which meant the special enrichment


of Caldilinea at 40–45 ◦C.B7 (Leptothrix cholodnii) and B8 (Acidothermus cellulolyticus) got

similar enrichment at 40–45 ◦C. Thereinto, B7 is a heterotrophic


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acterium, also majored in 40 ◦C and 45 ◦C, whose formed filamen-ous sheath can exhibit potential bioremediation [30].

These above-mentioned bacteria (B1, B3, B7 and B8) which werearticularly enriched at 40 ◦C and 45 ◦C, would take the responsi-ilities for the high COD removal in this temperature range (therst peak in Fig. 1). With the temperature increase, the micro-ial community would display evolution and the original dominanthermophilic community was eliminated and replaced by the newpecies with more competitive advantage at higher temperatureover 50 ◦C).

B9, B11, B13 and B14 which gradually enriched as the tem-erature increase, or only appeared at 50 ◦C and 55 ◦C (Fig. 3).hey all were identified as the thermophilic bacteria, whoselosest phylogenetic species were Geobacillus thermoglucosidasius,seudoxanthomonas taiwanensis, Geobacillus thermodenitrificansnd Rubellimicrobium thermophilum, respectively [31,32,33]. Thesepecies exhibited better adaptability at higher temperature andould contribute to the satisfactory COD removal at 50 ◦C and 55 ◦C

Fig. 1).Additionally, B12 was a denitrifier (Paracoccus denitrificans),

educing nitrite to N2O [32], and the anoxic growth agreed withts special enrichment in the biofilm lanes (Fig. 3). B14 (R. ther-

ophilum) was reported previously to be isolated from the colorediofilms [33], consistent with our study whose influent was alsoyeing wastewater.

The B5 (Nitrosomonas eutropha) was a kind of AOB which onlyan be observed at 35 ◦C and disappeared soon as the tempera-ure increase. The optimum growth temperature 35 ◦C agreed withhe previous study [34] and the NH3-N removal situation (Fig. 1).dditionally, the particular enrichment of B5 in the biofilm (rather

han the suspended sludge, Fig. 3), indicated the fillers can help toaintain nitrifying bacteria at relative higher temperature (35 ◦C,

ut helpless with much higher temperature).B6 was identified as Zunongwangia profunda which can secrete

arge quantity of EPS. Its high yield of EPS was confirmed to displayigh viscosity and great tolerance to high temperature [25], so thatan protect themselves and other bacteria nearby.

.3.2. The amoA genes quantification by real-time PCRThe quantification of amoA genes which has been extensively

pplied for the quantification of ammonia oxidizers, was furthernvestigated.

The density of amoA gene ranged from 0.87 × 103 to.45 × 106/mg sludge (Fig. 4), and the highest density was obtainedt the 35 ◦C level, especially for the biofilm. However, since theLSS (sludge) in the reactors (approximatively presented the total

acteria amount) decreased gradually as the temperature increasedFig. S2), the relative abundance of amoA genes (characterized byhe ratio of amoA/16S) would be more responsible for the nitri-cation ability of the sludge (Fig. 4). The abundance 0.06% in the

noculated sludge (about 30 ◦C) increased to ∼0.43% at the 35 ◦Cevel, and then decreased to 0.08% at 40 ◦C level.

The 40 ◦C owned even higher amoA abundance than the 30 ◦C,ince the abundance of other bacteria (total eubacteria) decreasedaster than AOB. It meant the activity of AOB at high temperature,.e. the thermotolerance of nitrifying ability has been enhanced.herefore, as the temperature increased (accumulation process),he AOB achieved further enrichment and could maintain its (rela-ive higher) abundance and nitrifying ability for a period of time.

Further, it can be noticed that the biofilm can help enrich moremoA genes (i.e. AOB), compared to the suspended sludge. This


esult also agreed with the previous DGGE analysis (the particularnrichment of B5, Fig. 3). Two reasons were probably responsible:1) the fillers facilitated the contact between microorganisms andxygen, in addition, (2) the fillers crashed into each other and cut

PRESStry xxx (2015) xxx–xxx 5

the bubbles into (more) micro-bubbles which promoted the oxygenutilization efficiency.

3.3.3. High-throughput sequencing (Miseq) for microbialcommunity analysis

High-throughput sequencing technology was applied to pro-vide further evaluation of the microbial community. The suspendedsludge at each temperature stage was further investigated, becauseaccording to the previous DGGE analysis (Fig. 3), the temperatureeffect plays more important role in microbial community variation,rather than the fillers effect. The top abundant genera (>0.4% for atleast one sample) in each sample were selected (a total of 30 generafor all 6 samples) and shown as base-10 logarithm, in the heatmap(Fig. 5).

The biodiversities, characterized by Shannon–Weiner index,were 4.92, 5.04, 4.64, 3.45, 3.06 and 2.94 for inoculated sludge(∼30 ◦C), 35 ◦C, 40 ◦C, 45 ◦C, 50 ◦C and 55 ◦C, respectively. In thegeneral view, the biodiversities decreased with the temperatureincreasing. It demonstrated that high temperature condition wouldeliminate many kinds of microorganisms, leading to lower biodi-versity (more simplex microbial communities) [13].

The cluster analysis demonstrated that the high similarityexisted between 50 ◦C and 55 ◦C, and the close relationship among35 ◦C, 40 ◦C and 45 ◦C (Fig. 5). It meant that as temperature increase,the great change of microbial community structure occurred at45–50 ◦C.

According to their abundance, many thermophilic genera,such as Rubellimicrobium and Pseudoxanthomonas began to beenriched at 50 ◦C, while others like Caldilinea was enriched from35 ◦C to 45 ◦C, but was eliminated gradually at higher temper-ature (50 ◦C). This is also consistent with the previous DGGEresults, which indicated that two groups of thermophilic micro-bial community—“lower and higher” were enriched and dominantat different temperature stages. In addition, the thermophilic genusSchlegelella [35] with high abundance in 50 ◦C and 55 ◦C, was furtherfound in miseq, rather than DGGE, whereas, it is on the con-trary for the genus Geobacillus (found in DGGE but not miseq),which meant these molecular technologies can be complemen-tary.

In this heatmap (Fig. 5), there was no AOB, but NOB (includeNitrospira and Nitrobacter) found whose abundance was >0.4%. Thepoor AOB abundance agreed with the real-time PCR results (Fig. 4).It is an indication that the NOB showed better thermostabilitythan AOB. And the abundance of NOB 0.83% (sum of Nitrospiraand Nitrobacter) decreased to 0.22% from 35 ◦C to 40 ◦C, and fur-ther exhibited sharp declination to 0.02% from 40 ◦C to 45 ◦C. Thevariation trend agreed with the pre-existing study which statedthe rapid declination of NOB activity was obtained at (from) 42 ◦C[34].

In summary, many kinds of thermophilic species or genera,belong to various classifications, were enriched with the grad-ually increased temperature. These thermophilic communitiesgradually evolved and acclimatized themselves to the chang-ing (increasing) selective pressure, and contributed to the CODremoval in the reactors with higher temperature. Further, the ther-mophilic community also played the role in NH3-N removal dueto their assimilation (directly), or led to a syngenetic growth withnitrifying bacteria (indirectly), which helped maintaining relativehigh NH3-N removal ability under the stress of high tempera-ture.

In this study, higher performance was achieved for dyeing


wastewater treatment (characterized by COD and NH3-N removal)at higher temperature condition (comparing to mesothermal con-dition), which mainly resulted from various thermophilic bacteriaenrichment in the sludge.



6 C. Li et al. / Process Biochemistry xxx (2015) xxx–xxx

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.4. The critical temperature effect on microbial community andiscussion on process of thermotolerant mechanism establishment

n the biological sludge

During the temperature gradually increase process, some criti-al temperature values (temperature change) should be concerned,hich greatly affected the microorganisms.

According to cluster analysis (Fig. 5), the microbial commu-ity structure showed great change from 45 ◦C to 50 ◦C. In other

◦ ◦


ords, the temperature critical value is “45 C|50 C” for the micro-ial community structure variation, which was mainly caused byifferent thermophilic community enrichment. Two taxa of ther-ophilic bacteria were enriched at different temperature range,

(to 16S gene) at each temperature stage by real-time PCR.

which can be grouped by “the precursor” at 35–45 ◦C level (lower)and “the successor” at 50–55 ◦C level (higher). The lower groupmainly includes Caldilinea (according to miseq), or B1, B3, B7 and B8(according to DGGE), whereas the higher contained genera Rubel-limicrobium and Pseudoxanthomonas (according to miseq), or B9,B11, B13 and B14 (according to DGGE). It demonstrated that thegreat change of dominant thermophilic community occurred at the“45 ◦C|50 ◦C”.

The “35 ◦C|45 ◦C” is the critical temperature of nitrifying bacte-◦ ◦


ria, among of which the AOB is “35 C|40 C” (based on the resultsof DGGE and real-time PCR), and the NOB is “40 ◦C|45 ◦C” (based onthe results of miseq), similar to previous study [34]. However, the“40 ◦C|45 ◦C” is another critical value for AOB in this study, accord-



C. Li et al. / Process Biochemistry xxx (2015) xxx–xxx 7

F ature

i

imth

Ef(

sape

mtadepFa4wt(ceT

ig. 5. The heatmap analysis of each suspended sludge sample at different tempernoculated sludge at ∼30 ◦C.

ng to real-time PCR (and the NH3-N removal situation), whicheant the thermophilic sludge (i.e. thermophilic community struc-

ure) could maintain the nitrifying ability (to a certain extend) atigher temperature.

The “40 ◦C|45 ◦C” is the critical temperature of the variation ofPS yield, since the EPS amount sharply increased to the maximumrom 40 ◦C to 45 ◦C, and then also sharply decreased over 45 ◦CFig. 2).

In summary, the biological thermotolerant mechanism in theludge mainly includes the thermophilic community enrichmentnd EPS variation, according to our study. Therefore, as the tem-erature increase, the whole process of thermotolerant mechanismstablishment in the sludge in MBBRs, is proposed as follows:

Before 40 ◦C as the temperature increase, the first group of ther-ophilic community (the precursor, with relative lower optimal

emperature) gradually enriches, which is dominant in the sludgend responsible for the main COD removal. Meanwhile, the abun-ance of nitrifying bacteria reduced, among of which AOB is firstliminated and then for NOB. The biofilm attached on the fillers canrotect nitrifying bacteria from being eliminated to a certain extent.rom 40 ◦C to 45 ◦C, the EPS amount increases sharply to becomenother contributor to the thermostability of the sludge (before0 ◦C, EPS slowly increases with the temperature), however, itould decrease after 45 ◦C. As the temperature further increases (to

he 50–55 ◦C stage), the second group of thermophilic community


the successor, with relative higher optimal temperature) whichan acclimatize themselves to higher temperature achieves morenrichment and replaces the first group (the precursor) gradually.he “two-stage enrichment” of different thermophilic communi-

stages (shown as base-10 logarithm of the abundance). “O” referred to the original

ties plays the key role in microbial community evolution and thethermotolerant mechanism establishment of the sludge.

The thermotolerant mechanism, as the temperature raise, is acomplex and integrated biological process, which is still lack of spe-cific theory. Although only the practical dyeing wastewater is used,the proposed thermotolerant mechanism process establishmentand the critical value of temperature, in this study, can providesome theoretical reference for the future research with differenttemperature in a more wide range (especially for dyeing effluenttreatment).

4. Conclusion

(1) During the temperature rising process from 30 ◦C to 55 ◦C, twopeak values of higher COD removal were obtained at 40 ◦Cand 50 ◦C, and the maximum nitrifying ability was observedat 35 ◦C and 40 ◦C, in the MBBRs under aerobic condition (DO∼2.0 mg/L).

(2) Two groups of thermophilic communities (“precursor and suc-cessor”) were enriched at different temperature stages, whichmainly conclude genera Caldilinea (from 35 ◦C to 45 ◦C) andgenera Rubellimicrobium and Pseudoxanthomonas (over 50 ◦C),respectively, i.e. the “45 ◦C|50 ◦C” is the critical temperature ofthe microbial community structure variation, especial for the


thermophilic community.(3) The process of biological thermotolerant mechanism estab-

lishment was discussed as temperature increasing, which isprobably based on the “two-stage enrichment” of thermophilic


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[35] R. Fabian, R. Simone, J. Dieter, Thermotolerantpoly(3-hydroxybutyrate)-degrading bacteria from hot compost andcharacterization of the PHB depolymerase of Schlegelella sp. KB1a, Arch.



communities and the EPS variation (first increasing and thendecreasing) in the sludge.

cknowledgements

This work was supported by the Major Science and Technol-gy Program for Water Pollution Control and Treatment of ChinaNo. 2012ZX07101-003), the Natural Science Foundation of Jangsurovince of China (BK20140852), and A Project Funded by the Pri-rity Academic Program Development of Jiangsu Higher Education

nstitutions.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.procbio.2015.08.07

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